Ultrathin porous nanoscale membranes, methods of making, and uses thereof

ABSTRACT

A process for forming a porous nanoscale membrane is described. The process involves applying a nanoscale film to one side of a substrate, where the nanoscale film includes a semiconductor material; masking an opposite side of the substrate; etching the substrate, beginning from the masked opposite side of the substrate and continuing until a passage is formed through the substrate, thereby exposing the film on both sides thereof to form a membrane; and then simultaneously forming a plurality of randomly spaced pores in the membrane. The resulting porous nanoscale membranes, characterized by substantially smooth surfaces, high pore densities, and high aspect ratio dimensions, can be used in filtration devices, microfluidic devices, fuel cell membranes, and as electron microscopy substrates.

This application is a divisional of U.S. patent application Ser. No.11/414,991, filed May 1, 2006, and claims the benefit of U.S.Provisional Patent Applications Ser. Nos. 60/675,963 and 60/782,001filed Apr. 29, 2005, and Mar. 14, 2006, respectively, which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to porous nanoscale membranes,particularly ultrathin porous nanoscale membranes, methods of making themembranes, and uses thereof.

BACKGROUND OF THE INVENTION

The separation of molecules in biological fluids is a basic procedureimportant in clinical diagnosis and disease treatment, and the mostelemental device for solute separation is a porous membrane filter.Thus, a revolutionary advance in filter technology has the potential toimpact many areas of human health. Typical filter materials are made aswoven matrices of plastic or cellulose polymers. Filters manufactured inthis manner naturally contain a wide distribution of pore sizes and thesmallest pores will eventually clog with small molecules of thefiltrate. The abundance of small pores and large filter thicknesses arethe two major sources of resistance to flow across membrane filters(Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters4:283-287 (2004)).

Two types of nanofabricated membranes have been previously developed formolecular separations. In the first type, molecules pass throughchannels with nanoscale diameters and lengths between 6 μm and 60 μm.Channel-type filters have been assembled by impregnating mesoporousalumina or polycarbonate host membranes with silica (Yamaguchi et al.,“Self-assembly Of A Silica-surfactant Nanocomposite In A Porous AluminaMembrane,” Nature Materials 3:337-341 (2004)) or gold (Lee et al.,“Electromodulated Molecular Transport In Gold-Nanotubule Membranes,” J.Am. Chem. Soc. 124:11850-11851 (2002)), and by the selective removal ofsilicon dioxide (SiO₂) from corrugated assemblies of silicon andpolysilicon (Martin et al., J. Controlled Release 102:123-133 (2005)).Channel-type membranes have been used successfully to separate small (˜1nm) molecules from proteins (˜5 nm) (Yamaguchi et al., “Self-assembly OfA Silica-surfactant Nanocomposite in a Porous Alumina Membrane,” NatureMaterials 3:337-341 (2004)), and to slow the diffusion of proteins fordrug delivery applications (Martin et al., “Tailoring Width ofMicrofabricated Nanochannels To Solute Size Can Be Used To ControlDiffusion Kinetics,” J. Controlled Release 102:123-133 (2005)). Whilethe large thickness of channel-type membranes provides the mechanicalstability needed for practical applications and large-scale separations,flow resistance (Tong et al., “Silicon Nitride Nanosieve Membrane,” NanoLetters 4:283-287 (2004)) and diffusion time increase directly withmembrane thickness. Novel aspects of channel membranes, such as thesub-Fickian movement of molecules constrained to move in single file(Wei et al., “Single-file Diffusion of Colloids in One-DimensionalChannels,” Science 287:625-627 (2000)), can be expected to slow fluxfurther. Long channels also create a lag between the initialintroduction of a mixture and the appearance of a species at thebackside of the membrane. For small macromolecules (˜1 nm) this delaycan be several hours (Yamaguchi et al., “Self-assembly Of ASilica-surfactant Nanocomposite in a Porous Alumina Membrane,” NatureMaterials 3:337-341 (2004)). Thus, while the use of channel-typenanomembranes for steady separations is possible, their use in rapidseparation procedures seems unlikely.

The issue of transport efficiency is addressed by a second type ofnanoporous membrane where the membrane is roughly as thick (˜10 nm) asthe molecules being separated. As an array of molecularly sized holes ina molecularly-thin plane, this type of membrane achieves a structurallimit that can help maximize filtration rates (Tong et al., “SiliconNitride Nanosieve Membrane,” Nano Letters 4:283-287 (2004); Chao et al.,“Composite Membranes From Photochemical Synthesis of Ultrathin PolymerFilms,” Nature 352:50-52 (1991)). In published work, however, such anultrathin membrane has been fabricated only by individually drilling 25nm pores in a 10 nm thick silicon nitride membrane using an ion beam(Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters4:283-287 (2004)). To provide mechanical integrity, this ultrathinmembrane was suspended over 5 micron holes patterned in a much thickerunderlying membrane. Large scale production of this type of membrane isimpractical because the pore drilling procedure is expensive andextremely slow (˜20 hours/cm²) (Tong et al., “Silicon Nitride NanosieveMembrane,” Nano Letters 4:283-287 (2004)). This method of manufacturewould not lend itself to a commercially useful filtration membrane.

The implementation of micro fluidics technology in small-scale proteinseparation and detection devices would benefit from the development ofan electrically switchable nanofluidic filter. Such a filter would allowthe construction of purification systems that can be electronicallytuned to capture proteins with sizes specified by a user. In the case ofa protein that is too dilute for detection or analysis, a dynamic filtercould be programmed to first concentrate by trapping the protein assolute flows past, and then release the protein into an analysischamber. There are several notable examples of switchable filters in therecent literature. In one example the charge on the membrane is activelymanipulated to repel like-charged species from entrance into pores(Schmuhl et al., “SI-Compatible Ion Selective Oxide Interconnects withHigh Tunability,” Adv. Mater. 16:900-904 (2004); Martin et al.,“Controlling Ion-Transport Selectivity in Gold Nanotubule Membranes,”Adv. Mater. 13:1351-1362 (2001)). In other examples, an electro-osmoticflow is established that drives solute charges past the membrane (Kuo etal., “Molecular Transport through Nanoporous Membranes,” Langmuir17:6298-6303 (2001)). These filters were made by composite assemblyprocedures that would be costly to integrate into a mass production. Theresulting filters are also thick, requiring species to pass throughmicron-long channels before emerging as filtrate. Large filterthicknesses are a major sources of resistance to flow-across membranefilters (Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters4:283-287(2004), and precludes the use of these designs in rapidseparation applications.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method of producinga porous nanoscale membrane. This method includes the following steps:applying a nanoscale film to one side of a substrate, the nanoscale filmcomprising a semiconductor material; masking an opposite side of thesubstrate; etching the substrate, beginning from the masked oppositeside of the substrate and continuing until a passage is formed throughthe substrate, thereby exposing the film on both sides thereof to form amembrane; and simultaneously forming a plurality of spaced pores in themembrane.

A second aspect of the present invention relates to a nanoporoussemiconductor membrane exposed on opposite sides thereof and having anaverage thickness of less than 500 nm, wherein said nanoporous membraneis prepared according to a process according to the first aspect of thepresent invention

A third aspect of the present invention relates to a nanoscalesemiconductor membrane exposed on opposite sides thereof, having anaverage thickness of less than about 500 nm, and having a plurality ofpores extending between the opposite sides thereof, wherein one or bothof the opposite sides are substantially smooth.

A fourth aspect of the present invention relates to a filter deviceincluding at least one nanoscale membrane according to the second orthird aspects of the present invention. The filter device preferablyincludes a support having a passage extending between opposite surfacesof the support, wherein the at least one nanoscale membrane is bound toor positioned on the support, with the at least one nanoscale membraneconfronting the passage.

A fifth aspect of the present invention relates to a microfluidic flowdevice that includes the filter device according to the fourth aspect ofthe present invention.

A sixth aspect of the present invention relates to a dialysis machinethat includes a filter device according to the fourth aspect of thepresent invention. The dialysis machine preferably contains multiplefilter devices of the present invention, each having a plurality of themembranes.

A seventh aspect of the present invention relates to a method offiltering nanoscale products. This method includes the steps of:providing at least one nanoscale membrane according to the second orthird aspects of the present invention; and passing a fluid, containingone or more products to be filtered, through the at least one nanoscalemembrane, whereby objects larger than the maximum pore size areeffectively precluded from passing through pores of the at least onenanoscale membrane.

An eighth aspect of the present invention relates to a device forcarrying out a biological reaction that includes: a substrate having oneor more wells, each having a bottom and one or more sidewalls, whereinthe bottom includes a nanoscale semiconductor membrane having an averagethickness of less than about 500 nm, and having a plurality of poresextending between the opposite sides thereof; and a biological reactionmedium in the one or more wells, and two or more reactants individuallyselected from the group of compounds and organisms.

A ninth aspect of the present invention relates to a method ofperforming a biological reaction. This method includes the steps of:providing a device according to the eighth aspect of the presentinvention; carrying out the biological reaction in the one or morewells, thereby forming an end product; and passing spent biologicalreaction medium from the one or more wells through the nanoscalemembrane, wherein the end product is either passed through the nanoscalemembrane or retained in the one or more wells.

A tenth aspect of the present invention relates to a method of screeningan agent for its activity on a cell. The method includes the steps of:providing an agent; performing a method according to the ninth aspect ofthe present invention by introducing the agent into the biologicalreaction medium that contains the cell; and analyzing the cell or theend product to identify the activity of the agent on the cell.

An eleventh aspect of the present invention relates to a fuel cell thatincludes at least one membrane according to the second or third aspectsof the present invention. A fuel cell of this type preferably includes amembrane that selectively passes positively charged ions.

The method of producing ultrathin nanoscale membranes of the presentinvention achieves a reproducible and cost-effective approach for theproduction of membranes that will be commercially useful in filtration,electron microscopy, nanobioreactor applications, and fuel cells. Inparticular, because the semiconductor materials are rendered porous in astep that allows all pores simultaneously to form during the annealingprocess, the pore density and size can be controlled by manipulating theannealing conditions and the thickness of the deposited semiconductormaterial. Moreover, because of the use of sacrificial layers on one orboth sides of the semiconductor film that is to form the final membrane,it is possible to prepare membranes with substantially smooth surfaces.This type of local smoothness is a useful feature for filters to be usedin many filtering applications.

Although the etching process takes 6 hours for a 500 nm thick siliconwafer, the etching process does not need to be continually monitoredand, therefore, is not time consuming. The process could very easily beautomated. Because the technique etches to a planar etch stop (at thesubstrate/first sacrificial film interface), fields of view approaching5,000×5,000 μm² of uniform thickness can be produced, which are ordersof magnitude larger than the viewable area produced with othertechniques. Perhaps the most unique feature of this process is itsconsiderable parallelism, producing up to several hundred porousnanoscale membranes in a single etching step. With other methodsdescribed in the literature, a researcher could easily spend a full daypreparing just a few samples, and these processes require fullattention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one embodiment of making a porousnanoscale semiconductor membrane of the present invention. References tosilicon and the crystal structure of the silicon are by way of exampleonly.

FIG. 2 illustrates the backside masking of elements so as to facilitateformation of an etched passage through the substrate (i.e., to exposethe membrane) as well as a trench in the substrate to allow for removalof an individual membrane. The masking patterns show variation acrossthe substrate, as different membrane sizes were optimized. All of themasks can be made substantially the same.

FIG. 3 illustrates a single surface etching cell used to ensure thatetchant is only exposed to the backside of the wafer. The solution isheated indirectly through the baseplate (shown) or through the sidewall,and the temperature is controlled using feedback from a thermocouplesubmerged in the solution (i.e., inside the etching cell).

FIGS. 4A-B illustrate a system where a membrane is bound in a device ora casing, thereby essentially creating a filter. In this system, theouter diameter can be adapted for use in a range of devices that utilizethe filters.

FIG. 5 is a schematic illustrating one embodiment for serial separationof proteins. The arrangement will allow the isolation of proteins intochambers based on protein size. Continuous flow will concentrateproteins before valves opens and drain individual compartments. Activecontrol of nanomembrane porosity (see FIG. 7) would allow a flexiblechromatography system to isolate proteins from a variety of bio fluids.

FIG. 6 illustrates a counter current flow cell incorporating anultrathin pnc-semiconductor membrane. These extremely high efficiencymembranes are particularly well suited to this separation technique,where continuous flow prevents accumulation of species near the membranethat can slow diffusion. This geometry also allows for high volumeseparation, as the carrier solution is not required to pass through themembrane, only the small product needs to diffuse across the barrier.

FIG. 7 shows active membrane switching methodologies. A porous membraneseparates two solutions A and B, and the switching voltage is eitherapplied directly to the membrane M relative to A/B or across themembrane from point A to point B.

FIGS. 8A-B illustrates a silicon wafer formed with a plurality of wellsthereon, each of which can be used to carry out a biological reactionbetween two or more agents and/or organisms. FIG. 8A illustrates a planview, and FIG. 8B illustrates a side elevational view.

FIGS. 9A-D illustrate the physical properties of pnc-Si membranes. FIG.9A shows refractive index dispersion curves of high optical densitysilicon films before (b, 15 nm a-Si) and after (a, 15 nm nc-Si)crystallization (729° C., 30 second anneal), determined by spectroscopicellipsometry. Dispersion curves for crystalline silicon (d, CrystallineSi) and CVD grown a-Si (c, CVD a-Si reference) are also plotted forreference. FIG. 9B is an AFM scan over the edge of a membranetransferred to a polished quartz window, which confirms the 15 nmthickness and minimal roughness of a pnc-Si membrane. FIGS. 9C-D areoptical micrographs of a 15 nm thick pnc-Si membrane at equilibrium,i.e., 0.0 pounds per square inch (“psi”), and with 15.0 psi of backpressure, demonstrating the remarkable strength of the ultrathinmembrane material.

FIGS. 10A-E depict 7 nm and 3 nm nc-Si membranes formed usingsacrificial 20 nm SiO₂ films. FIG. 10A is an interference contrastoptical micrograph of the released 3-layer 7 nm membrane (which showssimilar structure to a homogeneous oxide membrane). FIG. 10B illustratesthat dissolution of the oxide layers in buffered oxide etchant yields asubstantially flat 7 nm nc-Si membrane. FIG. 10C is an interferencecontrast optical micrograph of the planar 7 nm nc-Si membrane formedupon dissolution of the oxide layers in buffered oxide etchant. Theflatness of this membrane was measured to be less than 10 nm, and theroughness was less than 0.5 nm across a similar 200 μm membrane. FIG.10D is a plan view dark field TEM image of the 7 nm nc-Si film. Onlythose crystals with the proper crystalline orientation to supportelectron diffraction appear in this image. FIG. 10E shows a 3 nm thick400 μm wide membrane.

FIGS. 11A-B are plan view TEM test images of commonly available latexspheres. These test images confirmed the high electron transparency ofnc-Si/SiO₂ membranes and demonstrated that high resolution images can betaken through this structure. The background texture is nc-Si in themembrane.

FIGS. 12A-B depict a comparison of bright field and dark field images ofa 20 nm thick nc-Si film after RTA only. The bright field image contrastis due primarily to density differences, while the dark field patternsare formed by electron diffraction from crystalline material.

FIGS. 13A-C are TEM dark field images of nc-Si films with differentthicknesses. No clear nanocrystal size dependency is observed relativeto thickness, but the density of nanocrystals appears to increase forthicker films.

FIGS. 14A-B are plan view TEM images of 7 nm thick nc-Si films depositedwith the optimized a-Si sputtering conditions. The dark spots in theimages are holes in the film. Considerably more holes appear in the filmthat was both rapid thermal annealed (“RTA” or “RTP”) and furnaceannealed.

FIGS. 15A-C illustrate the tunability of pnc-Si membrane pore size. Byvarying the temperature at which the silicon film is crystallized, porediameter can be controlled. The maximum (cut-off) pore size and porosityincrease with annealing (RTA) temperature from 715° C. (FIG. 15A) to729° C. (FIG. 15B) to 753° C. (FIG. 15C), as illustrated in thehistograms that plot total pore area (left plot) and total number ofpores (right plot) available for molecular transport at each porediameter. The TEM image used to generate each histogram is included onthe center of the figure.

FIGS. 16A-C illustrate molecular separation and transport rates acrosspnc-Si membranes. The passage of two fluorescent species (labeledproteins or free dye) from a 3 μL source mixture through pnc-Simembranes was monitored simultaneously on two channels of a fluorescencemicroscope. The membrane edge was imaged from below and the lateralspread of fluorescent material was monitored to determine permeationthrough the membrane (FIG. 16B). An experimental image taken immediatelyafter the application of the source solution is also displayed, showingthe sharp fluorescence edge of the source solution behind the membrane(FIG. 16C).

FIGS. 17A-C show that using a membrane according to FIG. 15A, i.e.,Membrane A, highly efficient separation of BSA and free dye was observedthrough a membrane over 6.5 minutes. A plot of the fluorescenceintensity 50 microns from the membrane edge was generated from a timeseries of images (FIG. 17A). Intensities were normalized to the centervalue of the membrane in the first frame for each channel. The finalfluorescence image of each channel is shown in FIGS. 17B-C.

FIG. 18 shows that using a membrane according to FIG. 15B, i.e.,Membrane B, a 3-fold separation of proteins BSA (MW˜67 kD) and IgG(MW˜150 kD) was observed using the method described above. The largersize cutoff of this membrane, i.e., Membrane B, enables greater than 15×increase in the transport of BSA relative to the membrane of FIG. 15A,i.e., Membrane A.

FIG. 19 shows that the diffusion rate of dye through Membrane A, i.e.,the membrane according to FIG. 15A, was benchmarked relative to acommercial dialysis membrane with a 50 kD cutoff (50× larger than thedye MW). Because the 3 μL of source dye quickly depletes, the transportrate is calculated as the initial slope of the transport curve. Thedialysis membrane should be highly permeable to this 1 kD dye, yettransport is greater than 9× more rapid in the pnc-Si membrane.Concentration of the dye was measured by 558 nm absorption at each timepoint.

FIGS. 20A-C show separation experiments. The three panels demonstrateseparation of the three pairs of neighboring molecules in Table 1,referenced in Example 6. Each panel shows fluorescence images from themicroscope set-up (as illustrated in FIG. 16A) with the membrane and itscontained material visible on the right of the image. Average horizontalintensity profiles are shown adjacent to each image. FIG. 20A shows theseparation of dye and BSA using the membrane of FIG. 15A. FIG. 20B showsthe separation of BSA and IgG using the membrane of FIG. 15B. FIG. 20Cshows the separation of IgG and IgM using the membrane of FIG. 15C.

FIG. 21 shows the transport for fluorescently labeled IgG, diffusing inmicroscope chamber experiments (see, e.g., FIG. 16A). Consistent withthe expectation that nanomembranes carry intrinsic negative charge,increasing the ionic strength of the solvent enhances the rate ofdiffusion through membranes. Protein transport is doubled with 10×concentrated PBS buffer.

FIGS. 22A-C are gel electrophoresis experiments showing that pnc-Simembranes demonstrate a clear cut-off when filtering high concentrationbrain bovine extract (“BBEC”). The experiment in FIG. 22A employedNanosep columns. The experiment in FIG. 22B employed a commercialdialysis membrane. The experiment in FIG. 22C employed pnc-Si membranesaccording to the present invention. L is the ladder standard, C is thecontrol, R is the retentate, and F is the filtrate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to ultrathin nanoscale membranes,methods of making those membranes, and their use in macromolecularfiltration applications, nanobioreactors, and as a support for materialsduring electron microscopy.

According to one aspect of the present invention, the invention relatesto a method of making porous nanoscale semiconductor membrane.

The semiconductor material used to prepare the membrane can be anysuitable semiconductor material or combinations thereof, whether mixedas alloys or prepared as multilayered structures. Suitable semiconductormaterials include, without limitation, undoped silicon or germanium,p-doped silicon or germanium, n-doped silicon or germanium, or asilicon-germanium alloy. Exemplary p-doped silicon or germanium include,without limitation, those containing (CH₃)₂Zn, (C₂H₅)₂Zn, (C₂H₅)₂Be,(CH₃)₂Cd, (C₂H₅)₂Mg, B, Al, Ga, and In dopants. Exemplary n-dopedsilicon or germanium include, without limitation, those containing H₂Se,H₂S, CH₃Sn, (C₂H₅)₃S, SiH₄, Si₂H₆, P, As, and Sb dopants. The dopantscan be present in any suitable amount. Exemplary alloys of thesematerials include, silicon and germanium alloys in amounts of up toabout 10% by weight of germanium, as well as mixtures of thesematerials, and semiconductor materials based on Group III elementnitrides.

These semiconductor materials are preferred, because they are believedto behave in the same manner as undoped silicon with regard to theirability to crystallize. Thus, an amorphous film of a semiconductormaterial can be crystallized, as described below, to form ananocrystalline or polycrystalline membrane that is characterized bypores that pass through the nanoscale membrane. It should be appreciatedby those of skill in the art that any other materials that possess anintrinsic capability of crystallizing in this manner can likewise beused to form a porous nanoscale membrane of the present invention.

The method of preparing the membrane generally involves the steps ofapplying a nanoscale film to one side of a substrate, where thenanoscale film is formed of a semiconductor material as described aboveand also masking an opposite side of the substrate. These steps can beperformed in either order. After masking and applying the nanoscalefilm, the substrate is etched, beginning from the masked opposite sideof the substrate and continuing until a passage is formed through thesubstrate, thereby exposing the film on both sides thereof to form amembrane. The nanoscale film is rendered porous by annealing in a mannerthat achieves simultaneous formation of a plurality of randomly spacedpores in the film or membrane.

These steps are described in greater detail in association with theschematic illustration of FIG. 1. (Although FIG. 1 refers to amorphoussilicon and nanocrystalline silicon, consistent with the abovediscussion of semiconductors, it should be appreciated that suchreferences are by way of example only.)

The substrate to be used to support the nanoscale membranes ispreferably a substrate that can be easily masked and etched so that amembrane can be formed. Silicon is a preferred example of one suchsubstrate. Using standard 4, 6, 8, or 12 inch silicon wafers as thesupport, upwards of about 1500, 500 μm×500 μm, of the nanoscalemembranes can be formed on its surface depending on the desired lineararea to be occupied by the membrane. Using silicon as the example,silicon contains a naturally occurring thermal oxide layer. To simplifythe procedures used in preparing and forming the films of the presentinvention, this thermal oxide layer is preferably removed entirely fromone side of the substrate and partially removed (i.e., during themasking procedure) from the opposite side of the substrate, in bothinstances using a buffered oxide etchant (“BOE”) such as, but notlimited to, “buffer HF improved” etchant (Transene Company Inc., DanversMass.). This is illustrated in Steps A and B, respectively, of FIG. 1.

The masking step is preferably performed by forming an array of elementsthat will dictate the manner in which the unprotected substrate will beetched. According to one embodiment, illustrated in FIG. 2, each elementis composed of a square hole surrounded by a square outline. Thisallowed the formation of membrane structures, and also cut a trencharound each membrane so that samples could be individually removed fromthe wafer. Other configurations can, of course, be utilized withoutdeparting from the scope of the invention. When the feature sizes on themask are fairly large, they can be printed directly on film using a highquality laser printer. A film of this sort can be mounted on a glassmask blank and used as a standard photolithography mask. The backsideSiO₂ can be patterned with standard photolithography, followed by an ˜10min soak in 4:1 BOE to transfer the pattern into the oxide layer. Thephotoresist can be removed with acetone.

To the side of the substrate that lacks the oxide layer, a film of thesemiconductor material is applied to the substrate. In a preferredembodiment, the semiconductor film is preferably applied betweenapplications of sacrificial oxide films (i.e., one sacrificial filmapplied to the substrate, and the other sacrificial film applied to thesemiconductor film, thereby sandwiching the semiconductor film).

The sacrificial oxide films can be applied to form any desired reliefpattern in the semiconductor film, or the oxide and semiconductor filmscan be applied in a manner that achieves substantially planar films thathave little variation in thickness or surface roughness (i.e., the filmsare locally smooth). Relief patterns can be formed by, e.g., etching thefront face of the substrate prior to application of the sacrificialfilm.

Preferably, the oxide film is one that is etched at a rate that is atleast an order of magnitude, more preferably two orders of magnitudeless than the rate at which the substrate is etched by a particularetchant. This allows for precise control of the etching process;essentially, the etching process comes to a halt once the sacrificialoxide layer is encountered. This is illustrated in Step D of FIG. 1.

Any oxide film that resists etching (under conditions that are used toetch the substrate) can be used as the sacrificial film. Exemplary oxidefilms include, without limitation, silicon dioxide, titanium oxide, tinoxide, aluminum oxide, zinc oxide, hafnium oxide, and tantalum oxide.Based on testing of oxide membranes alone, it was expected that a 10 nmfilm of sputtered oxide would be sufficient to protect thenc-semiconductor layers from the etchant, although such membranes cannotmechanically support themselves alone. To build in additional tolerance,however, 20 nm sacrificial oxide films became standard for all nc-Simembrane fabrication and transmission electron microscopy imagingexperiments as described in the Examples.

Application of the oxide films and semiconductor films can be carriedout using any approach that allows for control of the film thickness.Exemplary approaches include, without limitation, radio-frequency (“RF”)magnetron sputtering, low pressure chemical vapor deposition (“CVD”),plasma enhanced CVD, thermal chemical growth, RF sputtering, DCsputtering, thermal evaporation, electron beam evaporation, andelectroplating. Of these, RF magnetron sputtering and electron beamevaporation are preferred. The conditions employed during the filmapplication steps can be selected according to the desired filmcomposition and thickness, as is known by those of skill in the art. Theconditions employed during the application can also be altered duringthe course of the application step, thereby achieving strata within thefilms that have varying properties. Exemplary condition changes include,without limitation, pressure changes, plasma density changes, plasmapower changes, temperature changes, gas composition changes, and sourcematerial changes.

With regard to the application of silicon dioxide and amorphous siliconfilms, the use of a high quality variable angle spectroscopicellipsometer (VASE) and a full wafer compatible atomic force microscope(AFM) allowed for reproducible and stable characterization of the filmsto be performed. This allowed for optimization of the conditionsutilized with RF sputtering procedures that were employed in theExamples described herein. Basically, SiO₂ films were reliably preparedat 750 W RF power, 15 mTorr chamber pressure, 6.0 sccm argon flow, and7.6 sc cm oxygen flow, where the films exhibit very low rms roughness(at most about 0.27 nm) and film thickness is dictated by the depositiontime. Amorphous silicon films were prepared at 150 W RF power, 15 mTorrchamber pressure, and 10.0 sccm argon flow. As with the oxide films, thedeposited thickness of a-Si was found to be a linear function of time.The use of other oxide films and other semiconductor films can beoptimized for the particular equipment that is employed.

Films that lack an intentional relief pattern are described herein asbeing substantially smooth or locally smooth. These terms are both usedto refer to the planar nature of the film surface(s), which is indicatedby a surface roughness of less than about 1 nm, preferably less thanabout 0.5 nm, most preferably less than about 0.4 nm, 0.3 nm, or 0.2 nm.

The porous nanoscale semiconductor membranes of the present inventioncan be prepared in any desired submicron thickness, but preferably lessthan 500 nm. In some embodiments, where the membranes are used inpressurized environments, porous semiconductor membranes of betweenabout 100 to about 500 nm, or about 150 to about 400 nm, or about 150 toabout 250 nm are preferred. In other embodiments, where the membranesare used in low pressurized environments or non-pressurizedenvironments, the membranes can be less than about 100 nm, morepreferably less than about 90 nm, about 80 nm, about 70 nm, or about 60nm, even more preferably less than about 50 nm, about 40 nm, about 30nm, or about 20 nm. In some embodiments, membranes of between about 2 toabout 25 nm are preferred. Particularly preferred are membranes that areless than about 10 nm thick, including those than are between about 1 nmto about 9 nm, about 2 nm to about 8 nm, about 2 nm to about 5 nm, andabout 6 nm and about 10 nm thick. Porosity and pore sizes of thesemembranes are described hereinafter.

Regardless of the materials selected, once the films have been formed,the semiconductor film is preferably annealed prior to any etchingprocedures. This is illustrated in Step C of FIG. 1. Annealing can beperformed after etching in some circumstances, particularly where theetch process removes only a limited amount of the total mass of thesubstrate. In other words, where the integrity of the etched substratewill not be altered during annealing, annealing of the semiconductormembrane (the released multilayered film) can be carried out afteretching. Otherwise, the annealing is preferably performed on thesemiconductor film prior to etching so as to minimize strain applied tothe membrane.

The annealing process can be carried out using known procedures forannealing semiconductor materials. Basically, these annealing processesafford sufficient heating of the semiconductor and sufficient dwell time(i.e., temperature and duration) so as to foster crystal growth.Preferably the annealing is performed in a manner that achievessubstantially uniform crystal growth, where the average crystal sizesare in the nanometer range, more preferably less than about 100 nm,about 90 nm, or about 80 nm in size, even more preferably less thanabout 70 nm or about 60 nm or about 50 nm in size, most preferably lessthan about 40 nm, about 30 nm, about 20 nm, about 10 nm, or even as lowas about 2 nm in size.

Suitable annealing processes include, without limitation, furnaceannealing, rapid thermal annealing (“RTA”), laser annealing, andcombinations thereof. Excellent results have been obtained with RTAalone or in combination with furnace annealing. With respect to theconversion of amorphous undoped silicon to form a crystalline (i.e.,nanocrystalline) silicon, preferred temperature ranges are between about650° C. and about 900° C., more preferably about 715° C. and about 800°C.; and preferred annealing durations are between about 20 seconds andabout 2 minutes for RTA, or between about 15 minutes and about 2 hoursfor furnace annealing. Conditions for doped silicon and silicon alloyswill vary slightly. Conditions for germanium will be from about 450° C.to about 900° C., more preferably about 500° C. to about 700° C., usingsimilar times for RTA and furnace annealing. The annealing temperatureand duration is also limited by the selection of the substrate uponwhich the films are formed; the temperature and duration should beselected to avoid deformation of the substrate.

Any suitable etching solution that, as noted above, selectively etchesthe substrate over the sacrificial oxide films can be employed. Whileone, two, or three orders of magnitude difference in selectivity issuitable, a preferred etchant has approximately four orders of magnitude(10⁴) difference in selectivity between silicon/silicon dioxide. Thispreferred etchant, known as EDP, contains ethylenediamine, pyrocatechol,pyrazine, and water. This etchant has an SiO₂ etch rate of only 0.2nm/min, with a corresponding (100) silicon etch rate of about 1400nm/min. Other advantages of EDP is that it contains no metal ions (suchas potassium or sodium) that create serious contamination issues incleanroom facilities, and this etchant solution is also known to producefewer hydrogen bubbles during etching, leading to a smoother etch front.

One preferred form of the EDP solution is commercially available fromTransene Company, Inc. (Danvers, Mass.) under the label PSE300F (i.e.,“Preferential Silicon Etch 300—Fast”). This solution contains 1 Lethylenediamine, 320 g pyrocatechol, 320 mL water, and 6 g pyrazine.

Because EDP requires about 6 hours to etch through the substrate, andEDP would otherwise have consumed the thin sputtered films on the frontsurface if exposed to EDP for the full 6 hours, an etch cell was createdthat allows for submersion of the backside (i.e., masked side) of thesubstrate while allowing the film-deposited side of the wafer to remainunexposed to the etchant. This therefore avoids the need for thick oxidemasks, which would have been required for submersion of the entire waferin the etch cell.

As noted above, the etching process is carried out beginning from thebackside (i.e., masked side) of the substrate. This process isillustrated in Step D of FIG. 1. This process is preferably carried outin a etch cell 10 of the type illustrated in FIG. 3. Basically, astainless steel cylinder collar 12 is sealed over the backside of themasked substrate (i.e., masked Si wafer 16) using a nitrile O-ring 14mounted in a groove at the base of the cylinder. The O-ring allows atight seal to be formed against the wafer to be etched. A support wafer18 provides a planar support surface to the membranes when they arereleased from the substrate (i.e., when etching of the substrate iscompleted). The etching solution can be heated through the baseplate 20(or through the sidewalls of the etching cell) with, e.g., an electricalresistance heater, and a stable solution temperature can be maintainedwith feedback from a thermocouple. Magnetic stirring can also appliedthrough the baseplate, ensuring adequate mixing of the etchant. Acondenser (not shown) mounted to the top of the cell can be used toprevent excessive evaporation during the ˜6 hour etches. Using an etchcell of this type requires only ˜250 mL of EDP for a standard 4 inchsilicon wafer. This allows fresh solution to be used for every etch,promoting reproducibility.

Although there are several ways to design a tool that etches only asingle surface of a semiconductor wafer, the above-described design hasthe advantage of making very efficient use of the etching solution. The250 mL used for each etch is near its silicon saturation limit and canbe discarded after each use. Other techniques may involve protecting oneside of the wafer and submerging it in a much larger volume EDP bath. Alarger volume is not usually disposed of after each etch, due toexpense, and therefore each successive etch will see a solution withdifferent properties.

Following etching, all that remains across the expanse of the etched pitis the sacrificial film-protected membrane. This membrane is exposed onboth sides, but at this stage it is not porous due to the presence ofthe non-porous sacrificial films. In a final step, the sacrificial filmsare removed from both sides of the crystalline (nanoporous)semiconductor nanoscale film. Removal of the sacrificial oxide films iscarried out using an etchant that selectively etches oxide films overthe semiconductor film/membrane. Suitable etchants include, withoutlimitation, buffered oxide etchant solutions and hydrofluoric acidetchant solutions. Exemplary buffered oxide etchant solutions include,without limitation, about 6 to about 25 wt % (more preferably about 6 toabout 10 wt %) HF (BOE contains HF and ammonium fluoride—concentrationsvary by manufacturer). Exemplary hydrofluoric acid solutions includeabout 5 to about 25 wt % HF, more preferably about 5-15 wt % HF.

Conditions employed for removal of the sacrificial oxide films includedipping the etched substrate in an appropriate etchant solution forsufficient amount of time to cause the oxide films to be substantiallyremoved from the underlying porous semiconductor nanoscale film. Typicaltimes for a 20 nm oxide film, at an oxide etchant temperature of about22° C., include between about 10 sec and about 8 minutes.

Upon removal of the sacrificial oxide films, the resulting porousnanoscale membrane is exposed on its opposite sides. Individualmembranes can be removed from the larger wafer given the masking patternutilized (described above). Alternatively, a whole wafer can bemaintained with a plurality of the membranes formed thereon.

The porous nanoscale membranes have thicknesses within the rangesdescribed above. Depending upon the annealing conditions that areemployed, the porosity and average pore sizes (including maximum poresize) can be controlled. By way of example, membranes having averagepore sizes of less than about 50 nm in diameter can be prepared. Moreparticularly, membranes can be prepared so as to tailor average poresizes within the range of about 25 to about 50 nm, about 20 to about 25nm, about 15 to about 20 nm, about 10 to about 15 nm, and about 2 toabout 10 nm. Pore densities between 10⁶-10¹² cm⁻² can be obtained.

Additional control over the pore size distribution can be achieved byslowly reducing the size of the as-formed pores by filling them in withanother material. For example, the RF magnetron sputtering process fordepositing amorphous silicon could be applied to the nanoporousmembrane. Depositing approximately 1 nm of amorphous silicon couldreduce the average pore diameter by as much as 2 nm. By carefullycontrolling this subsequent deposition, pore size distribution muchsmaller than those that can be formed directly, can be achieved.

The membranes are also characterized by a large surface area notpreviously obtained for semiconductor membranes. In particular, surfaceareas up to about 10 mm² have been obtained. In addition, membraneshaving lateral length to thickness aspect ratios greater than 10,000:1have been obtained. In certain embodiments, aspect ratios greater than50,000:1, 100,000:1, and 430,000:1 have been obtained. Given thestability of the films and the manufacturing procedures, it is expectedthat aspect ratios of up to 1,000,000:1 can be achieved.

In one embodiment, the nanoscale membrane is formed of a semiconductormaterial and further includes a coating of a metal at least partiallycovering one side of the membrane. Metals can be partially coated ontothe membrane using DC or RF sputtering, thermal evaporation, electronbeam evaporation, electroplating, or wet chemical deposition. Suitablemetals include, without limitation, gold, silver, copper, platinum,aluminum, chromium, titanium, tungsten, lead, tin, palladium, and alloysof these metals.

As noted above, the ultrathin porous nanoscale membranes of the presentinvention can be used to form a filter device for filtration of anymaterial, either particulates or dissolved solids, entrained in fluids.In its simplest form, the filter device of the present inventionincludes at least one of the porous nanoscale membranes.

In its simplest form, therefore, the filtering of nanoscale products canbe carried out by passing a fluid, containing one or more products to befiltered, through the membrane, whereby objects larger than the maximumpore size are effectively precluded from passing through pores of themembrane.

Fluids that can be subject to filtration can be any gas or liquid,including aqueous solutions. According to one embodiment, the fluid tobe filtered is a biological fluid. Exemplary biological fluids include,without limitation, saliva, blood, serum, cerebral spinal fluid, mucosalsecretions, urine, and seminal fluids.

Products or materials to be filtered can include, without limitation,proteins, viruses, bacteria, colloidal nanoparticles, organic molecularsystems, inorganic nanoparticles, dissolved ions, drugs, dyes, sugars,aqueous salts, metals, semiconductor particles, and pharmaceuticalcompounds. The materials to be filtered can also be a charged species(either negatively or positively charged).

According to one embodiment, the filter device further includes asupport having a passage extending between opposite surfaces of thesupport, wherein the at least one nanoscale membrane is bound to thesupport, with the at least one nanoscale membrane confronting thepassage. The support can be the un-etched semiconductor support to whichthe membranes are attached. Alternatively, if one or more of themembranes are removed, e.g., from the etched wafer, the membranes andtheir accompanying supports can be embedded in a device that providesthe membrane(s) in one or more passages for filtering materials from afluid capable of passing through the membrane.

Examples of such devices are shown in FIGS. 4A-B. This type of devicecan be in the form of an adapter 30, 40 (having an inlet I and an outletO, with the support and membrane 35, 45 being positioned on or securedto the adapter such that the passage through the support is in fluidcommunication with the inlet and outlet), where the adapter is designedfor installation in existing filtration equipment. A microfluidic flowdevice, a dialysis machine, or a fuel cell can include the filter deviceaccording the present invention.

With regard to microfluidic flow devices, the ability to activelycontrol the permeability of semiconductor membranes will allow theconstruction of dynamic microfluidic devices for protein purificationand analysis. Among these applications, tunable microchromatographysystems and microfluidic systems that concentrate proteins beforereleasing them to an analysis chamber can be utilized.

In one embodiment, the filter device includes two or more nanoscalemembranes having different maximum pore sizes, wherein the two or morenanoscale membranes are serially positioned relative to the direction offluid flow with a membrane having a smaller maximum pore size beingdownstream of a membrane having a larger maximum pore size.Alternatively, the filter device includes three or more nanoscalemembranes serially positioned as described above. Depending upon thematerials to be filtered, any number of membranes can be used to provideserial filtration in this manner. Serial filters of this type areillustrated in FIG. 5.

The results presented herein demonstrate faster filtration and bettersize selectivity than existing commercial filters, and with simpleintegration of the membranes into microfluidic systems, serialfiltration devices will allow for the rapid fractionation and assay ofblood proteins or other fluids containing mixed populations of proteinsand other compounds. Such a device could replace expensive andtime-consuming electrophoresis assays currently used in clinical bloodlabs (Williams et al., Biochemistry in Clinical Practice, Elsevier, N.Y.(1985), which is hereby incorporated by reference in its entirety) witha rapid assay requiring a finger-prick's worth of blood (20 uL).

The ability to dynamically adjust pore sizes or to control permeabilityto particular species with an on/off switch will allow for production ofmicrofluidic devices that first concentrate and then release a proteininto a reaction chamber or sensor in lab-on-a-chip applications. Anadjustable pair of pnc-Si membranes can define the entry and exit wallsof a protein isolation chamber in an on-chip purification system, andbecause the pore sizes would be tunable with a voltage setting, the samesystem can be used to purify different proteins from cocktails. Furtherassembling more than two of these membranes in series will allow theconstruction of an adjustable microfluidic chromatography system tofractionate a protein cocktail an a user-defined fashion (FIG. 5).

This work with pnc-Si membranes represents the first use of ultrathinplanar-type nanomembranes for size-based molecular separations. First,the separation of serum molecules, albumin and IgG, suggests theintegration of pnc-Si membranes with microfluidic blood analyzers orantibody purification systems. In fact, several membranes may bearranged in series to develop microfluidic systems that fractionatecomplex protein mixtures. Most significant is the improved rate oftransport achieved with pnc-Si membranes. The diffusion measurementsrecorded transport of 156 nmol/cm² per hr for Alexa dye. This rate ismore than one order of magnitude faster than those reported forchannel-type membranes for similarly-sized molecules (Yamaguchi et al.,“Self-Assembly of a Silica-Surfactant Nanocomposite In a Porous AluminaMembrane,” Nature Materials 3:337-341 (2004), which is herebyincorporated by reference in its entirety), and greater than 9× fasterthan measurements through 50 kD cut-off cellulose dialysis membranes(described in Example 5). The porous nanocrystalline semiconductor-basedplatform opens several avenues for commercial developments includingscalable production of membranes, straightforward integration intomicrofluidic devices, surface modifications using well-establishedsilane chemistries, and pore size reduction though vacuum deposition ofinorganic films (Tong et al., “Silicon Nitride Nanosieve Membrane,” NanoLetters 4:283-287 (2004), which is hereby incorporated by reference inits entirety). Importantly, the demonstrated mechanical strength ofthese ultrathin membranes will allow the construction of large-scaledialysis systems and facilitate the use of ultrathin membranes inpressurized filtration devices at the macro and micro-scale.

In a further embodiment, illustrated in FIG. 6, the filter deviceincludes first and second conduits positioned in a substantiallyparallel arrangement, with the at least one nanoscale membranepositioned between the first and second conduits. This allows fordissolved materials and possibly fluids to pass between the first andsecond conduits as limited by the pore size of the at least onenanoscale membrane. The direction of fluid flow for the first and secondconduits can be in the same direction or in the opposite direction.

In another embodiment, the filter device includes an electrode coupledto or positioned adjacent the at least one nanoscale membrane. This typeof filter device can be used as a gated membrane. The device can includea first electrode positioned on one side of the nanoscale membrane and asecond electrode positioned on an opposite side of the nanoscalemembrane, and optionally a third electrode coupled to the membraneitself (which has a metal contact thereon, as described above). Thisembodiment is illustrated in FIG. 7. Upon introduction of an electrolytesolution across the nanoscale membrane, the first and second electrodesare capable of applying a voltage across the electrolyte solution. Thethird electrode can be used to charge the membrane per se. During use ofthis type of filter device, charged species can be filtered by applyinga voltage differential across the membrane.

Gated membranes may separate species via any one of three transportmechanisms: Fickian diffusion, ion migration, and electro-osmosis. Eachelectrical switching approach relies on the influence of theelectrochemical double layer (the space-charge region created in asolution near a charged surface) on molecular transport throughnanoscale pores. As pore size approaches the double layer thickness anda substantial fraction of the pore volume is filled by the space chargeregion, its influence can be used to actively control transport. Thedouble layer thickness is approximated by the Debye length κ⁻¹=(3.29zC^(1/2))⁻¹, where κ⁻¹ is in nm and C is the molar concentration of theelectrolyte (Kuo et al., “Molecular Transport Through NanoporousMembranes,” Langmuir 17:6298-6303 (2001), which is hereby incorporatedby reference in its entirety). Therefore, the double layer thicknessvaries from about 30 nm to about 1 nm in aqueous solutions with 0.1 mMto 100 mM ionic concentrations, respectively. The field polarity andmagnitude within the double layer is determined by the surface chargedensity on the pore wall, a function of surface chemistry and solutionpH.

Martin and coworkers have developed a process in which commerciallyavailable track-etched polycarbonate porous membranes are coated with aprecise thickness of gold (Martin et al., “Controlling Ion-TransportSelectivity in Gold Nanotubule Membranes,” Adv. Mater. 13:1351-1362(2001), which is hereby incorporated by reference in its entirety).Track-etched filters are characterized by a monodisperse distribution ofstraight cylindrical pores that extend completely through a 10 micronthick membrane. Gold deposition on the pore walls reduces the porediameters from ˜30 nm to ˜3 nm, enhancing the influence of the doublelayer, and creating a highly conductive membrane material. A voltage canthen be applied to this membrane, raising (lowering) its potentialrelative to solutions A and B, in the geometry depicted in FIG. 7. Thiscauses a decrease (increase) in the electron density in the membranethat perturbs the field intensity of the double layer, therebymodulating the membrane permeability. Clear shifts in diffusion ratesfor charged ions have been shown with other membrane constructions.

Kuo and coworkers have demonstrated the use of similar polycarbonatetrack-etched membranes with 15 nm pore size to inject species when avoltage is applied across it (Kuo et al., “Molecular Transport throughNanoporous Membranes,” Langmuir 17:6298-6303 (2001), which is herebyincorporated by reference in its entirety). The long narrow pores inthese membranes tend to have a net positive surface charge on the porewalls that attracts negative counter ions in the double layer.Electro-osmotic flow occurs when a voltage is applied between solutionsA and B in the geometry of FIG. 7, inducing flow of mobile counter ionsalong the length of the pores or channels. This effect becomes strongeras the double layer occupies more of the chamber volume and this fillingcan be adjusted by changing the ionic strength of the solutions. Flowbetween solutions A and B can be increased or stopped entirely byapplying a relatively high voltage across the nanoporous membrane.

Schmuhl et al. have developed a novel membrane material that can beintegrated on a silicon microelectronic platform (Schmuhl et al.,“SI-Compatible Ion Selective Oxide Interconnects With High Tunability,”Adv. Mater. 16:900-904(2004), which is hereby incorporated by referencein its entirety). Using a 1 micron thick silicon nitride membrane with1.2 micron diameter pores filled with porous silica to achieve aneffective pore size of ˜3 nm, Schmuhl et al. have demonstrated thatswitching behavior in the flow of both cations and anions is achieved byapplying a voltage across the membrane between solutions A and B.Switching occurs at a fairly low 2V and there is a clear ‘off’ state atzero voltage. The switching geometry is similar to that used forelectro-osmotic switching, but Schmuhl has demonstrated that this effectis dominated by direct flow of ions in the applied electric field(electrophoresis). In this manner, positive or negative charges can beselectively pumped across the membrane by applying appropriate voltage.Again, this selectivity is possible because the double layer occupiesmost or all of the nanoscale pore volume. All of the switchingapproaches should be replicable using the nanoscale porous membranes ofthe present invention, but diffusion rates should be enhanced.

Metal nanoscale membranes of the present invention, preferably thoseformed of platinum, palladium, or a palladium alloy, can be used in fuelcells of the type described, e.g., in U.S. Pat. Nos. 6,319,306 and6,221,117 to Edlund et al., both of which are incorporated by referencein their entirety. In this embodiment, the nanoscale membraneselectively passes positively charged species such as hydrogen.

The filter device according to the present invention can further includeone or more capture molecules within the pores or on one or bothsurfaces of the membrane. The capture molecules are selected from thegroup of antibodies, nucleic acids (RNA or DNA), polypeptides, molecularprobes with specific or non-specific affinity for a target molecule, andcombinations thereof.

The filter device according to the present invention can also includeone or more non-binding, screening, or repulsive molecules tethered tothe membrane within the pores. The non-binding, screening, or repulsivemolecules can include, without limitation, Teflon, polyethylene glycols,and other organic molecules.

Regardless of the type of molecule tethered to the surface of themembranes or within the pores, standard glass coupling chemistry can beused. Basically, the semiconductor material that forms the membrane isprovided with a thin oxide coating thereon. This process can occurnaturally in air, can be accelerated with slightly elevated temperaturesand humid environments, and can also be accomplished by exposing thesamples to an oxygen plasma at low (20-30° C.) to moderate (100-400° C.)temperatures. The molecules to be tethered can then be bound using anyof a variety of coupling strategies.

The available strategies for attaching the one or more capture moleculesor the one or more non-binding, screening, or repulsive moleculesinclude, without limitation, covalently bonding the one or moremolecules to the surface of the semiconductor membrane, ionicallyassociating the one or more molecules with the surface of thesemiconductor membrane, adsorbing the one or more molecules onto thesurface of the semiconductor membrane, or the like. Such association canalso include covalently or noncovalently attaching the one or moremolecules to another moiety (of a coupling agent), which in turn iscovalently or non-covalently attached to the surface of thesemiconductor membrane.

Basically, the oxidized and hydrolyzed surface of the semiconductormaterial is first functionalized (i.e., primed) with a coupling agentwhich is attached to the surface thereof. This is achieved by providinga coupling agent precursor and then covalently or non-covalently bindingthe coupling agent precursor to the surface of the semiconductormembrane. Once the semiconductor surface has been primed, the one ormore molecules to be bound are exposed to the primed semiconductorsurface under conditions effective to (i) covalently or non-covalentlybind to the coupling agent or (ii) displace the coupling agent such thatthe one or more molecules covalently or non-covalently binds directly tothe oxidized semiconductor surface. The binding of the one or moremolecules to the semiconductor membrane is carried out under conditionswhich are effective to allow the one or more molecules to remainavailable for interacting (e.g., binding, repelling, etc.) with speciesto be filtered.

Suitable coupling agent precursors include, without limitation, silanesfunctionalized with an epoxide group, a thiol, an aldehyde, NHS ester,or an alkenyl; and halide containing compounds.

Silanes include a first moiety which binds to the surface of thesemiconductor structure and a second moiety which binds to the tetheredmolecules. Preferred silanes include, without limitation,3-glycidoxypropyltrialkoxysilanes with C1-6 alkoxy groups,trialkoxy(oxiranylalkyl)silanes with C2-12 alkyl groups and C1-6 alkoxygroups, 2-(1,2-epoxycyclohexyl)ethyltrialkoxysilane with C1-6 alkoxygroups, 3-butenyl trialkoxysilanes with C1-6 alkoxy groups,alkenyltrialkoxysilanes with C2-12 alkenyl groups and C1-6 alkoxygroups, tris[(1-methylethenyl)oxy]3-oxiranylalkyl silanes with C2-12alkyl groups,[5-(3,3-dimethyloxiranyl)-3-methyl-2-pentenyl]trialkoxysilane with C1-6alkoxy groups, (2,3-oxiranediyldi-2,1-ethanediyl)bis-triethoxysilane,trialkoxy[2-(3-methyloxiranyl)alkyl]silane with C1-6 alkoxy groups andC2-12 alkyl groups,trimethoxy[2-[3-(17,17,17-trifluoroheptadecyl)oxiranyl]ethyl]silane,tributoxy[3-[3-(chloromethyl)oxiranyl]-2-methylpropyl]silane, andcombinations thereof. Silanes can be coupled to the semiconductormembrane according to a silanization reaction scheme shown in FIG. 9A ofPCT Publication No. WO/2002/068957, which is hereby incorporated byreference in its entirety.

Halides can also be coupled to the semiconductor membrane according tothe reaction scheme set in FIG. 9B of PCT Publication No.WO/2002/068957, which is hereby incorporated by reference in itsentirety.

Thereafter, the one or more molecules are bound to the semiconductormembrane according to the type of functionality provided by the one ormore molecules. Typically, the one or more molecules are attached to thecoupling agent or displace to coupling agent for attachment to thesemiconductor membrane in aqueous conditions or aqueous/alcoholconditions.

Epoxide functional groups can be opened to allow binding of amino groupsaccording to the reaction scheme set forth in FIG. 10A of PCTPublication No. WO/2002/068957, which is hereby incorporated byreference in its entirety. Epoxide functional groups can also be openedto allow binding of thiol groups or alcohols according to the reactionscheme set forth in FIGS. 10B-C, respectively, of PCT Publication No.WO/2002/068957, which is hereby incorporated by reference in itsentirety.

Alkenyl functional groups can be reacted to allow binding of alkenylgroups according to the reaction scheme set forth in FIG. 10D of PCTPublication No. WO/2002/068957, which is hereby incorporated byreference in its entirety.

Where a halide coupling agent is employed, the halide coupling agent istypically displaced upon exposing the primed semiconductor membrane toone or more molecules (as described above), which contain alcohol groupsas the semiconductor-binding groups. The displacement can be carried outaccording to the reaction scheme set forth in FIG. 10E of PCTPublication No. WO/2002/068957, which is hereby incorporated byreference in its entirety.

Where the one or more capture molecules contain two or moretarget-binding groups, it is possible that the target-binding groups mayalso interact and bind to the primed surface of the semiconductormembrane. To preclude this from occurring, the primed poroussemiconductor membrane can also be exposed to a blocking agent. Theblocking agent essentially minimizes the number of sites where the oneor more capture molecules can attach to the surface of the semiconductormembrane. Exposure to the blocking agent can be carried out prior toexposing the primed surface of the semiconductor membrane to the capturemolecule or simultaneous therewith. The blocking agents can bestructurally similar to the capture molecules except that they lack atarget-binding group or the blocking agents can be simple end-cappingagents. By way of example, an amino acid alkyl ester (e.g., glycinemethyl ester, glycine ethyl ester, 3-alanine methyl ester, etc.)blocking agent can be introduced to an epoxide-functionalizedsemiconductor structure surface as shown in FIG. 10A of PCT PublicationNo. WO/2002/068957, which is hereby incorporated by reference in itsentirety, except with the amino group of glycine opening the epoxidering and covalently binding to the coupling agent.

Another aspect of the present invention relates to a device for carryingout a biological reaction. The device includes a substrate comprisingone or more wells, each having a bottom and one or more sidewalls,wherein the bottom comprises porous nanoscale membrane of the presentinvention. One embodiment of such a device is illustrated in FIGS. 8A-B.The device also includes a biological reaction medium in the one or morewells, and two or more reactants individually selected from the group ofcompounds and organisms. Each well can have a bottom made of a distinctnanoscale membrane or all of the wells can be equipped with the sameporous nanoscale membrane.

Suitable compounds can include, without limitation, any one or more ofproteins, colloidal nanoparticles, organic molecular systems, inorganicnanoparticles, dissolved ions, drugs, sugars (saccharides orpolysaccharides), aqueous salts, metals, semiconductors, pharmaceuticalcompounds, nucleic acids, enzymes, lipids, carbohydrates, andpolypeptides.

Suitable organisms can include, without limitation, a cell line, aprimary isolated cell or mixed population of cells, one or morebacteria, a virus, a protoplast, fungus, a parasite, and combinationsthereof.

In one embodiment, the membrane includes a cell surface receptor, anantibody, a ligand, a biological reagent, or a combination thereof, onthe surface of one or both sides of the membrane or within the pores ofsaid membrane.

In use, a device of this type can be used to carry out a biologicalreaction in the one or more wells. The biological reaction, if itoccurs, forms an end product that can be detected or measured (i.e.,quantified). By passing spent biological reaction medium from the one ormore wells through the nanoscale membrane, the end product is eitherpassed through the nanoscale membrane or retained in the one or morewells. Its presence in the well (retentate) or in the filtrate can bedetected by an immunoassay or nucleic acid detection assay. Exemplaryimmunoassays include, without limitation, ELISA, radioimmunoassay,gel-diffusion precipitation reaction assay, immunodiffusion assay,agglutination assay, fluorescent immunoassay, protein A immunoassay, andimmunoelectrophoresis assay. Exemplary nucleic acid detection assaysinclude, without limitation, polymerase chain reaction, Northernblotting, Southern blotting, ligase detection reaction, and LAMPdetection assay. Other detection procedures using color-changing dyes orthe like can also be employed.

One embodiment of this biological reaction relates to a method ofscreening an agent for its activity on a cell. This method involvesintroducing an agent into the biological reaction medium, which includesthe cell, and then analyzing the cell or an end product to identify theactivity of the agent on the cell. The same detection proceduresemployed above can be utilized to detection a cellular response (i.e.,upregulation of receptor or cellular protein or other secondarymessenger), or direct examination of the cells can be carried out.Assays for targeted cellular responses can be incorporated with thebioassay and filtration procedures described herein.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1 Formation of Nanocrystalline Silicon Membranes

Pnc-Si membranes were fabricated by the procedure outlined in FIG. 1. A500 nm thick layer of SiO₂ on both sides of a silicon wafer was firstgrown by placing it in a semiconductor tube furnace at 1000° C. for 2hours in an ambient of oxygen and steam. On the backside of the wafer,the SiO₂ was patterned using standard photolithography techniques toform an etch mask for the membrane formation process. The etch mask foreach membrane included a square-shaped opening centrally positionedwithin a square border (up to 120 masks have been formed on a singlesilicon wafer). The front oxide layer was then removed by exposing thesurface to 4:1 BOE, for 10 minutes, in an etching cell similar to thatused for EDP etching, and a high quality three layer film stack (20nm-SiO₂/15 nm-amorphous silicon/20 nm-SiO₂) was RF magnetron sputterdeposited on the front surface. The a-Si layer was sputtered at achamber pressure of 15 mTorr in Ar with a target power density of 0.4W/cm², yielding a deposition rate of 3.4 nm/min. The SiO₂ layers werereactively sputtered from a silicon target at a chamber pressure of 15mTorr, with a (3:4) Ar:O₂ gas flow ratio, and a target power density of1.8 W/cm², yielding a deposition rate of 10.7 nm/min. The depositionrecipe is well characterized and capable of depositing films with +/−1%thickness accuracy and surface roughness less than 0.5 nm.

Crystallization of very thin amorphous silicon films, forming highquality nanocrystals with well-defined size has been previouslydemonstrated (Grom et al., “Ordering and Self-Organization InNanocrystalline Silicon,” Nature 407:358-361 (2000), which is herebyincorporated by reference in its entirety). To form the pnc-Simembranes, the substrate was briefly exposed to high temperature (715°C.-770° C. for 30 sec) in a rapid thermal processing chamber,crystallizing the amorphous silicon into a nanocrystalline film. Thepatterned wafer backside was then exposed to the highly selectivesilicon etchant EDP (Reisman et al., “The Controlled Etching of SiliconIn Catalyzed Ethylenediamine-Pyrocatechol-Water Solutions,” J.Electrochem. Soc. 126:1406-1415 (1979), which is hereby incorporated byreference in its entirety), which removes the silicon wafer along (111)crystal planes until it reaches the bottom silicon dioxide layer of thefront side film stack.

The etching procedure began by exposing the masked backside of thepatterned wafer to a 50:1 (hydrofluoric acid:water) solution for 2minutes to remove any native oxide in the unprotected areas. 250 mL ofEDP was then heated to 80° C. in a pyrex beaker while the wafer wasmounted in the etching cell. This preheating was performed to preventthe temperature from overshooting the set value during the ramp up tothe temperature set point. The preheated EDP was then poured into theetch cell (see FIG. 3) and the solution was heated to 105° C. withstirring. After the temperature stabilized at 105° C., the systemremained in a constant equilibrium state for the duration of the etch.Under these conditions, an etch rate of approximately 1.4 μm/minute wasobtained, completing a 500 μm etch in about 6 hours.

After etching was complete, the EDP was removed from the cell and thewafers were carefully rinsed in deionized water. Exposing the threelayer membrane to 15:1 buffered oxide etchant (BOE) for 60 sec at 22° C.removed the protective oxide layers leaving only the freely suspendedultrathin pnc-Si membrane. A final rinse in ethanol or pentane wastypically used to reduce the surface tension while drying the membranes.

This process has been used to fabricate square membranes as thin as 3 nmand as thick as 500 nm; and square membranes as small as 10 μm×10 μm andas large as 3 mm×3 mm. This etch has also been used to outlineapproximately 120 samples (3.5 mm×3.5 mm) that can be easily removedfrom the wafer after the fabrication process is complete, and usedindividually for molecular separation experiments.

Example 2 Formation of Additional Nanocrystalline Silicon Membrane

The procedure described in Example 1 was used to form a 7 nm a-Si layersandwiched between two 20 nm layers of SiO₂ using sputter deposition;the silicon was then annealed at 950° C. for 30 sec to form a nc-Silayer. After release, a wrinkled membrane similar to other oxidemembranes was formed (FIG. 10A). However, after the removal of thesacrificial SiO₂, with a 25 second dip in buffered oxide etch (BOE), anextremely flat 7 nm ne-Si membrane was produced (see FIGS. 10B-C). Thisdramatic shift from compressive to tensile stress was unexpected, yet isindicative of the volume contraction that occurs when a-Si iscrystallized (Miura et al., Appl. Phys. Lett. 60:2746 (1992), Zachariaset al., Journal of Non-Crystalline Solids 227-230:1132 (1998), which arehereby incorporated by reference in their entirety). Since the oxideremains compressively stressed after annealing and is much thicker thanthe a-Si, the overall film stress is compressive in the initial wrinkledfilm. However, after the oxide is removed, the nc-Si layer is free toreturn to a tensile state. This two step membrane release process isalso very robust with a yield greater than 80% in initial 7 nm processtests. FIG. 10D is a dark field TEM image of a 7 nm nc-Si film showingcrystals with dimensions similar to the film thickness, confirmingprevious reports (Tsybeskov et al., Mat. Sci. Eng. B 69:303 (2000),which is hereby incorporated by reference in its entirety). To test thephysical limits of this process, the 3 nm silicon membrane in FIG. 10Ewas fabricated with a width:thickness aspect ratio exceeding 100,000:1.It is believed that this is the thinnest, high aspect ratio membraneever reported and it clearly demonstrates the capabilities of the singlesurface etching (“SSE”) process. It is possible that even thinnermembranes can be formed; no limit has yet been achieved.

To test the electron transparency of these membrane structures, 90 nmlatex spheres were deposited on a few of the three layer sandwichsamples with a 7 nm oxide. As seen in FIGS. 11A-B, the membranes werevery transparent, the latex was easily imaged, and fine structure isclearly observable. Upon careful inspection, some silicon nanocrystalsare observable in the background texture.

In FIGS. 12A-B, a comparison of bright field and dark field TEM imagesis made for a 20 nm nc-Si layer that only received an RTA atapproximately 750° C. for 60 sec. The bright field mode is directlyanalogous to an optical microscope operating in transmission. Contrastis obtained primarily through density variations in the sample. In thisparticular image the grain structure is clearly visible but the degreeof crystallization cannot be readily determined. A dark field image isformed only by electrons that have diffracted from crystalline material.Therefore any bright spots in the image are nanocrystals. However, notall crystals are at the proper orientation to support diffraction, andtherefore the observed nanocrystals are assumed to be a small fractionof the total.

A comparison of films with substantially different thickness wasperformed to determine the effect of a-Si layer thickness in the lateralsize of the nanocrystals. For thin layers the nanocrystals were expectedto be spherical and for thicker films, considerably larger brick shapeshave typically been observed. The images in FIGS. 13A-C do not agreewith these previous observations and surprisingly seem to indicate thatno clear size dependence exists for films in this thickness range,although the nanocrystal aerial density does appear to increase withincreasing thickness. All images were taken at the same magnificationand are directly comparable. It is unclear why the current nc-Simaterial does not crystallize in the same manner as has been observed inthe past. This result is surprising, and illustrates the potentialimportance of plan view TEM studies in characterizing film structure inthis material system.

Images of two 7 nm thick nc-Si layers deposited high refractive indexa-Si sputtering conditions are displayed in FIGS. 14A-B. One film wastreated with a 750° C., 60 sec RTA only (14A), and the other had both a750° C., 60 sec RTA and a 1050° C., 10 min furnace anneal (14B). Allanneals were done in a nitrogen environment. Both images are dark fieldand were taken at the same magnification. As with the previous TEMimages, a substantial number of bright nanocrystals are observable,however there are also black spots in both images that had not beenobserved in other samples. These spots are actually holes in the film.In dark field, only properly aligned crystals appear bright, butamorphous and misaligned crystalline material will also scatter theelectron beam and produce a very weak background illumination. The darkspots in the images are the absence of this background, indicating thatno material is present. This interpretation also agrees with similarbright field images. It is also apparent from these images that thelonger furnace anneal promotes the formation of these features becausethe hole density is much higher in the left image and are either due tovoids in the film itself or they are regions of oxide formed during theannealing process that are removed when the barrier oxides are etchaway. It was later determined through testing that these holes areactually pores extending through the membrane.

Example 3 Physical Properties of Porous Nanocrystalline SiliconMembranes

In addition to TEM, several other characterization techniques have beenused to confirm the properties of the pnc-Si membranes according to thepresent invention. FIG. 9A shows refractive index dispersion dataobtained using spectroscopic ellipsometry for a 15 nm thick silicon filmafter deposition (a-Si) and after crystallization (pnc-Si) (Tompkins etal., “Spectroscopic Ellipsometry and Reflectometry—A User's Guide,”Wiley & Sons, Inc., New York, (1999), which is hereby incorporated byreference in its entirety). The sputtered a-Si has high optical density,comparable to microelectronic quality a-Si deposited with chemical vapordeposition (CVD), and exhibits a clear shift in optical properties aftercrystallization, with characteristic resonance peaks similar tocrystalline silicon (Palik, E. D. “Handbook of Optical Constants ofSolids,” (Academic Press, Orlando) pp. 547-569 (1985), which is herebyincorporated by reference in its entirety). This data is indicative ofhigh purity silicon films with smooth interfaces. It should also benoted that TEM images of the as deposited a-Si show no distinguishablevoids or crystalline features. To confirm the accuracy of thespectroscopic ellipsometry data, several membranes were transferred ontopolished quartz and atomic force microscopy (AFM) was used to measurethe step height of the membrane edge confirming the 15 nm thickness ofthe sample membrane and its highly smooth surface.

Another important characteristic of pnc-Si membranes is their remarkablemechanical stability. Membranes were mechanically tested using a customholder to apply pressure to one side of the membrane while an opticalmicroscope was used to monitor deformation. FIGS. 9C-D show opticalmicrographs of a 200 μm×200 μm×15 nm membrane as 1 atm (15 PSI is thelimit of the experimental fixture used) of differential pressure wasapplied across it for approximately 5 minutes. With no differentialpressure, the membrane is extremely flat (FIG. 9C), and at maximumpressure (FIG. 9D) the membrane elastically deforms but maintains itsstructural integrity throughout the duration of test. Unlike thinpolymer membranes (Jiang et al., “Freely Suspended NanocompositeMembranes As Highly Sensitive Sensors,” Nature Materials 3:721-728(2004), which is hereby incorporated by reference in its entirety),pnc-Si membranes exhibit no plastic deformation and immediately returnto their flat state when the pressure is removed. Pressurization testswere cycled three times with no observable membrane degradation. Theremarkable strength and durability exhibited by these membranes islikely due to their smooth surfaces (Tong et al., “Silicon NitrideNanosieve Membrane,” Nano Letters 4:283-287 (2004), which is herebyincorporated by reference in its entirety) and a random nanocrystalorientation that inhibits the formation and propagation of cracks.

Example 4 Pore Size Distribution in Porous Nanocrystalline SiliconMembranes

It was also determined that the pore size distribution in pnc-Simembranes can be controlled through adjustment of the rapid thermalannealing temperature during crystallization. Nanocrystal nucleation andgrowth are Arrhenius-like processes (Spinella et al., “Crystal GrainNucleation In Amorphous Silicon,” J. Appl. Phys. 84:5383-5414 (1998),which is hereby incorporated by reference in its entirety) that exhibitstrong temperature dependence above a threshold crystallizationtemperature of approximately 700° C. in a-Si (Zacharias et al., “ThermalCrystallization of Amorphous Si/SiO₂ Superlattices,” Appl. Phys. Lett.74:2614-2616 (1999), which is hereby incorporated by reference in itsentirety). Existing crystallization models (Zacharias et al.,“Confinement Effects In Crystallization and Er Doping of SiNanostructures,” Physica E. 11:245-251 (2001), which is herebyincorporated by reference in its entirety) fail to predict voidformation and must be extended to account for how volume contraction andmaterial strain lead to pore formation in ultrathin membranes.

To demonstrate pore size tunability, three wafers with 15 nm thickpnc-Si membranes were processed identically, according to the previouslydescribed methods, except for the annealing temperature. TEM images ofthese membranes shown in FIGS. 15A-C revealed that pore size and densityincrease monotonically with temperature, as samples annealed at 715° C.,729° C., and 753° C. have average pore sizes of 7.3 nm, 13.9 nm, and21.3 nm, respectively. A sample annealed at 700° C. exhibited nocrystallinity or voids, illustrating the strong morphological dependenceon temperature near the onset of crystallization. The tunability of poresize in this range makes pnc-Si membranes particularly well suited forsize selective separation of large biomolecules, such as proteins andDNA. Because pore area is a key metric for the discussion of moleculartransport through these membranes, histograms that identify the totalpore area available at each pore size are presented in FIGS. 15A-C. Poresize data was extracted directly from the TEM images using Scion imageprocessing software (Scion Corporation, Frederick, Md.).

Example 5 Protein Separation Using Porous Nanocrystalline SiliconMembrane

To demonstrate molecular separations with pnc-Si, two common bloodproteins of different molecular weight (MW) and hydrodynamic diameter(D), BSA (MW=67 kD, D=6.8 nm) and IgG (MW=150 kD, D=14 nm),fluorescently labeled with Alexa 488 and Alexa 546 (Molecular Probes,Eugene Oreg.), respectively, were chosen. Free Alexa 546 dye was alsoused as an additional low molecular weight (M=1 kD) species. The dyereacts with primary amines forming stable covalent bonds. Each specieswas twice purified with spin columns provided in the labeling kit.Protein concentration and degree of labeling was calculated by measuringabsorbances with a spectrophotometer using extinction coefficientsprovided by the dye manufacturer. This analysis showed that BSA waslabeled with eight moles of dye per mole of protein, while IgG waslabeled with three moles of dye per mole of protein. With the microscopeused for the above experiments, this yielded similar fluorescenceintensity for each species at the same concentration. In the separationexperiments, proteins were used at 1 μM, while free dye was used at 100μM to mimic the separation of proteins from higher concentration solutespecies as might occur in a buffer exchange or desalting application.

The passage of these species through the pnc-Si membranes, shown in FIG.16B, was monitored using real time fluorescence microscopy. In thissetup (FIG. 16A), a membrane and its supporting silicon wafer frame wasplaced on a glass slide with 50 μm silica spacers forming a thindiffusion chamber beneath the membrane. The chamber was first filledwith approximately 50 μL of clean phosphate buffered saline (“PBS”).Then, 3 μL of a fluorescent protein (1 uM) and/or dye (100 uM) in PBS,as described above, was added to the well above the membrane. An imageof the membrane edge was taken every 30 seconds in each of thefluorescent channels. The passage of each species through the membranewas observed as the spreading of fluorescence signal from the membraneedge. FIG. 16C shows a false color image of the membrane edgeimmediately after the mixture was added to the well. The membraneappears bright from the fluorescent species in the well above, and theuniform darkness beyond the membrane edge indicates that no fluorescentmolecules have passed through the membrane into the underlying chamber.

FIG. 17A shows the results of a membrane permeation experiment comparingBSA to free Alexa 546 dye using the membrane of FIG. 15A. The passage ofthe two fluorescent species (labeled BSA and free dye) from a 3 μLsource mixture through pnc-Si membranes was monitored simultaneously ontwo channels of a fluorescence microscope (FIG. 16B). The membrane edgewas imaged from below and the lateral spread of fluorescent material wasmonitored to determine permeation through the membrane. An experimentalimage taken immediately after the application of the source solution isalso displayed, showing the sharp fluorescence edge of the sourcesolution behind the membrane. The false color images (FIGS. 17B-C) showhow the fluorescence spreads after 6.5 minutes for each species and theadjacent graph (FIG. 17A) shows a quantitative comparison of thefluorescence intensity. From these results it is clear that dye passesfreely through the membrane while BSA is almost completely blocked.

There are several possible reasons why BSA was retained behind amembrane with maximal pore sizes more than twice as large as themolecule's hydrodynamic diameter. Charge-charge interactions betweenproteins and the membrane's native oxide layer, protein adsorption thatpartly obstructs pores, and uncertainty in the relationship betweenhydrodynamic dimensions and the physical size of proteins, could allcontribute.

FIG. 18 shows a similar experiment, performed as described above for theBSA experiment, where the permeability of IgG and BSA at 1 μMconcentration was compared using the membrane of FIG. 15B. In this case,BSA diffused through the membrane ˜3× more rapidly than IgG. Because themolecular diffusion coefficients for these molecules are within 25% ofeach other (Karlsson et al., “Electronic Speckle Pattern Interferometry:A Tool For Determining Diffusion and Partition Coefficients For ProteinsIn Gels,” Biotechnol. Prog. 18:423-430 (2002), which is herebyincorporated by reference in its entirety), the measured rate differenceclearly indicates that pnc-Si membranes hinder IgG diffusion relative toBSA diffusion. By more thoroughly optimizing pore sizes, one can expectto engineer pnc-Si membranes that can completely exclude IgG but permitBSA passage. However, even with the existing membranes, it should bepossible to enhance the separation by arranging several membranes inseries. It should also be noted that the plots of FIGS. 17A and 18 canbe quantitatively compared for BSA, demonstrating that the increasedcut-off size of membrane B (FIG. 15B) allows a 15× enhancement of BSAdiffusion relative to membrane A (FIG. 15A).

Given the hours-long passage-times of molecules through channel-typemembranes (Yamaguchi et al., “Self-Assembly of a Silica-SurfactantNanocomposite In a Porous Alumina Membrane,” Nature Materials 3:337-341(2004), which is hereby incorporated by reference in its entirety), itis significant that filtrate molecules appear downstream of pnc-Sifilters within minutes. To better quantify the transport through pnc-Simembranes, the fluorescence microscopy experiments were followed withbenchtop experiments in which one could remove and assay the Alexa 546dye that diffused across membrane A from a 100 μM startingconcentration. Dye diffusion through pnc-Si membranes was compared todiffusion through standard regenerated cellulose dialysis membranes.

Pnc-Si membranes were assembled on an aluminum support. The volume belowthe membrane was filled with 100 μL of clean PBS. 3 μL of 100 μMfluorescent dye in PBS was placed in the Si well above the membrane. Atthe time points shown in FIG. 19, the 100 μL, from beneath the membranewas removed. Absorbance was measured on a spectrophotometer to determinethe concentration of dye that had transported across the pnc-Simembrane. To measure dye transport across the 50 kD dialysis membrane,diffusion was measured across a comparable membrane surface area. Thedialysis membrane was pulled taut and sealed against the tip of amicropippette. 3 μL of 100 μM fluorescent dye in PBS was loaded into thepipette. The entire pipette was submerged into a microcentrifuge tubewith 100 μL of clean PBS. At the specified time points, the 100 μLvolume was removed and measured for absorbance. For both experiments,each time point is representative of unique experiments.

The results shown in FIG. 19 reveal that diffusion through pnc-Simembranes is over 9× faster than dialysis membranes with comparable sizeexclusion properties. The pnc-Si membranes exhibit an initial transportrate of 156 nmol/cm² per hr (FIG. 19) that rapidly slows as the 3 μLsource volume depletes, lowering the concentration gradient across thebarrier. A dialysis membrane with a 50 kD cut-off was chosen for thisexperiment, based on the excellent retention of BSA (66 kD) by membraneA of FIG. 15A. Remarkably, when this experiment was repeated on membraneC, i.e., FIG. 15C, for 1 hour, an increase of less than 10% in dyetransport was measured, despite porosities differing by 30 fold (0.2%vs. 5.7%). This indicates that dye transport is essentially unhinderedby the membranes according to the present invention, as porosities farlower than that of membrane A should theoretically allow greater thanhalf-maximal diffusion through an infinitely thin porous barrier (Berg,H. C., “Random Walks in Biology,” Princeton University Press, pp. 34-37(1993), which is hereby incorporated by reference in its entirety).Therefore, the observed greater than 9× increase in diffusion rate overconventional dialysis membranes is essentially the physical limit offree diffusion in this experiment.

Example 6 Protein Separation of Three Species Using Serial Separation

To test the capacity of pnc-Si membrane to filter blood proteins,fluorescent red and green-labeled bovine serum albumin (BSA), IgG, andIgM were created (Table 1), and diffusion through membranes wasmonitored using a fluorescence microscope.

TABLE 1 Molecules Used in Separation Studies Bovine Free SerumFluorecent Albumin IgG IgM Dye (BSA) antibody antibody MW ~1,000 ~66,000~150,000 ~970,000 (daltons) Experimental ~1 × 10⁻⁶ ~1 × 10⁻⁶ ~1 × 10⁻⁶~1 × 10⁻⁶ Concentration (M) Protein mass/ — ~0.07 ~0.15 ~0.97 volume(mg/ml) Hydrodynamic 1 nm ~3.5 nm ~7 nm ~14 nm radius (nm)

The membrane and its silicon support were positioned 50 μm from amicroscope coverslip using silica beads as spacers (see, e.g., FIG.16A). The channel between the coverslip and the membrane structure wasfilled with phosphate buffered saline. A 3 μl volume containing one redand one green fluorescently labeled species was then added to the top ofthe membrane. The microscope system imaged the arrival of fluorescentmolecules in the channel using a field-of-view that included one edge ofthe membrane. In this way early images contained a sharp intensitydifference at the membrane edge that diminished with time as fluorescentmolecules passed through the membrane.

Experimental results are shown in FIGS. 20A-C. Each of the three mainpanels of this figure compares the transport for two molecules ofneighboring molecular weight using one of the membranes of FIGS. 15A-C.In all three cases: IgM and IgG (FIG. 20C), IgG and BSA (FIG. 20B), andBSA and free dye (FIG. 20A), a membrane provided excellent separationbetween the two molecules after 11 minutes in the system. FIG. 20A showsnearly perfect exclusion of BSA and rapid passage of dye. This suggeststhat these membranes may be highly effective for the selective removalof small molecular weight compounds from larger blood proteins in renaldialysis applications. Protein separations in FIGS. 20B-C are excellentdespite the fact that only the membranes characterized in FIGS. 15A-Cwere available as candidates in testing. By engineering an array ofmembranes with different mean pore sizes, it is expected that membraneswith better selectivity for BSA and IgG will be found.

It is noteworthy that the mean size of the membranes is larger than thesize of the protein they appear to retain. For example IgG, with ahydrodynamic radius of 7 nm (Luby-Phelps et al., “Hindered Diffusion ofInert Tracer Particles in The Cytoplasm of Mouse 3T3 Cells,” Proc. Natl.Acad. Sci. 84(14):4910-3 (1987), which is hereby incorporated byreference in its entirety) is largely excluded from passage throughmembranes with a significant number of pores that should permit passage(>14 nm). The results suggest effective pore diameters that are smallerthan the physical measurements in TEM, and/or effective protein sizesfor filtration that are larger than the hydrodynamic radii determined insedimentation assays. One reasonable possibility for smaller pore sizesis that a surface oxide layer is present on membranes aftermanufacturing. Such layers naturally contain negative charges thatshould draw counter (positive) ions to the membrane surface in an ionicsolution. Given the nanometer dimensions of pores, positive chargescould dominate the interior of the pores and retard the passage ofnegatively charged species. Martin et al. have demonstrated thisprinciple for nanotubes under similar circumstances for small chargedmolecules (Martin et al., “Controlling Ion-Transport Selectivity in GoldNanotubule Membranes,” Adv. Mater. 13:1351-1362 (2001), which is herebyincorporated by reference in its entirety). Consistent with the proposalthat nanoscale ordering of charges could reduce the permeability ofpnc-Si membranes, faster passage of IgG using a higher ionic strengthbuffer was found (FIG. 21). The concentration of ions in this solutionshould significantly reduce the depth of organized charge (the debyelayer). With an isoelectric point of 7.2, the IgG molecule itself shouldbe neutral (as a temporal average) in PBS (pH 7.3), however the Alexadye that was used to follow proteins carries a charge of −2 in neutralpH. Because the charge on the protein will fluctuate, one might expectthe phenomenon to slow but not to block the labeled IgG. Thus, theresults obtained thus far are consistent with the idea that thepermeability of pnc-Si membranes can be tuned by manipulating surfacecharge.

Example 7 Comparison of Porous Nanocrystalline Silicon Membrane AgainstCommercial Protein Separation Filters

A pnc-Si membrane was compared to two commercially available proteinseparation filters, 100 kD Nanosep™ and 50 kD dialysis membrane, both ofwhich were obtained from their manufacturers (Pall Corporation, EastHills, N.Y.; and Spectrum Laboratories, Rancho Dominguez, Calif.,respectively). The proteins from each experiment were analyzed with gelelectrophoresis to determine size separation. In each gel, the left laneor column is the ladder standard (L), which identifies the location ofdifferent sized proteins. The smaller proteins migrate further down thegel. The column identified by (C) is the control, which contains all ofthe proteins in the extract for each experiment. (R) is the retentate,which are the larger proteins retained above or behind the filter media.(F) is the filtrate, which contains the proteins that passed through thefilter media.

In the Nanosep experiment (FIG. 22A), 2004 clarified BBEC (1.2mg/mL) wasspun using 100 kD cutoff Nanosep columns (Pall Life Sciences) for 5minutes at 14000 g. Samples were recovered from both top and bottom offilter, and the retentate was diluted to match the concentration offiltrate. The Nanosep columns retained proteins under the specified 100kD cutoff, and high losses were observed in comparison to the pre-spuncontrol. After removal of samples, a noticeable brown stain remained inthe membrane, most likely due to proteins trapped within the relativelythick membrane. 50 kD cutoff dialysis tubing (FIG. 22B) was used toseparate 1 mL BBEC (1.2 mg/mL). After 24 hours, the retentate wasremoved (since the filtrate was diluted into a large vessel, it couldnot be run on a gel). Very little protein below the specified cut-offwas filtered. With the pnc-Si filters (FIG. 22C), it was possible to usea much higher concentration of protein without clogging. 1.5 μL BBEC (6mg/mL) was separated using the pnc-Si membrane and a 50 mL PBSreservoir. Proteins greater than 50 kD remained in the retentate, whileproteins smaller than 50 kD became very dilute since they easilydiffused across the membrane into the filtrate. The pnc-Si membranesshowed very little loss of sample compared to Nanosep membranes.(L=Ladder, C=Control, R=Retentate, F=Filtrate)

Example 8 Voltage-Gated Nanoscale Membranes

One distinctive aspect of pnc-Si membranes is their molecular height. Bycontrast, the membrane pores used in previously reported switchingmethodologies discussed in the literature are approximately 1000× longerthan they are wide. This extreme aspect ratio limits the peak transportefficiency. Given the rapid diffusion of molecules through pnc-Simembranes, as demonstrated in the preceding Examples, the ultrathinmembranes according to the present invention should overcome thislimitation upon demonstration of strong switching behavior. Furthermore,the membranes are not limited to small ion transport, but should allowmacromolecules like proteins to be selectively transported (asdemonstrated above). The membranes are also created using an inexpensivefabrication on a silicon platform, which should enable their simpleintegration into microfluidic systems. Because these ultrathin porousmembranes are fundamentally different (material, aspect ratio,fabrication, etc.) than the membranes previously studied for activeswitching, it is expected that voltages will tend to move chargedspecies through the membrane and that surface charges will tend torestrict the passage of like-charged molecules. These principles will betested in the prospective experimental work described below.

Negative surface charge occurs naturally as oxide layers develop onsilicon surfaces during manufacturing. A voltage applied to pnc-Simembranes coated with conducting metals can induce additional negativecharge. Charged membrane surfaces should attract counter ions fromsolution into pores and limit entrance by similarly charged molecules.For conductive membranes, either positive or negative voltage can beapplied, and so positively charged macromolecules could be also berestricted. These behaviors will allow for the active control of theseunique membranes in microfluidic devices that separate and analyzeproteins.

In a first experiment, the extent to which the intrinsic permeability ofa pnc-Si membrane is determined by surface charge produced duringmanufacturing will be assessed. In a second experiment, the potential toactively manipulate the surface charge of pnc-Si membranes coated withconductive metals will be examined. In a third experiment,electrophoretic flows past pnc-Si membranes will be established. Toavoid ambiguous conclusions, the transport of well-defined quantum dotswill be examined (but results obtained with the quantum dots shouldconfirm the capability of achieving similar results with proteins andsmall charged molecules).

Experiment 1

It is known that oxide layers that naturally grow on pnc-Si membranesduring manufacturing will cause fixed surface charges that may affectprotein transport. These charges may explain why some proteins areapparently unable to pass through certain membranes that have asignificant numbers of pores larger than the protein's hydrodynamicradius. Demonstrating that charge dependent permeability is an intrinsicproperty of pnc-Si membranes will be an important baseline for studiesinto active control of transport.

The general strategy will be to monitor the passage of fluorescentspecies with controlled charges using a fluorescent microscope. With theassumption that the membranes carry net negative charge aftermanufacturing, the expectation is that negatively charged species willbe blocked (or pass more slowly) through pnc-Si membranes. Because theamine and carboxyl groups found on proteins have pKAs around 3 and 8respectively, one approach would be to examine the diffusion of proteinsthrough pnc-Si membranes in electrolyte (Phosphate Buffered Saline)solutions of different pHs. Neutralizing the negatively chargedmolecules should hasten transport, however protein sizes or membranecharge may be affected by pH changes, and so results obtained with thisprocedure may be challenging to interpret.

To avoid complications in interpretations, amine and carboxylderivatized quantum dots will be used as test species instead ofproteins. This model system is ideal because, unlike with proteins,charge and particle size are independently controlled for quantum dots.Quantum dots with amine and carboxyl surface coatings are commerciallyavailable from Evident Corp. The company offers these surfacechemistries, as well as neutral surfaces, on 25 nm (hydrodynamic)diameter quantum dots with a variety of fluorescence emissions. Pnc-Simembranes with the majority of pores between 20 and 40 nm can be readilymanufactured and used in these experiments. Transport of positively,neutral, and negatively charged particles through the membranes will bemonitored using the same flow chambers used to monitor the separation offluorescent proteins by pnc-Si membranes. The expectation is that COO—quantum dots will pass slower through pnc-Si membranes despite being thesame size as neutral and amine particles.

With this experimental design, there are additional experiments whichwill reinforce these interpretations. First, the charges on bothpositive and negatively charged quantum dots will be neutralized usingcarbimimide reactions with acetate and aminomethane, respectively. Theexpectation is that neutralized versions of COO— particles and amineparticles will diffuse through pnc-Si membranes at the same rate asneutral particles.

Experiment 2

In a second type of experiment to confirm the role of membrane surfacecharge in species transport, the ionic strength of the solute containingcharged quantum dots will be increased by increasing salt (KCl)concentrations from 0 to 0.5 M. The expectation is that as ionicstrengths increase, the depth of the debye layer that shields surfacecharge will grow smaller than pore diameters, and so the differencesbetween the rates of transport for charged and uncharged species willdiminish.

Regardless of whether the intrinsic charge on pnc-Si membranes issufficient to affect the transport of charged species, it should bepossible to intentionally charge pnc-Si membranes provided they arecoated with a conducting material. The active manipulation of chargefrom an exogenous source provides the best opportunity to activelymanipulate the selectivity of pnc-Si membranes.

The experiments conducted under this study are similar to those outlinedabove except that the pnc-Si membranes used will be manufactured withgold coatings. Pnc-Si membranes will be coated by two techniques. First,coating will occur using standard evaporation techniques. Evaporationshould produce a uniform gold layer on one side of pnc-Si membranes.Because the interior surface of pores should be uncoated, it is unclearif this coating approach would be as effective as a technique thatcoated all surfaces with gold. One such technique is electroless goldplating—a chemical reduction process that involves the catalyticreduction of gold ions in solution and the subsequent deposition of theions into a surface layer. To ensure efficient deposition it may benecessary for the surface to carry surface bound amines, in which casethe silicon surfaces of pnc-Si membranes can be pretreated with aminosilane. The transformation of the pnc-Si surfaces will be confirmed bySEM. If coating is found to significantly reduce pore diameters, largerpore membranes will be used for the starting material and/or theexposure of the surfaces to gold ions will be adjusted to limit thegrowth the deposited layer.

The rate of passage of charged, uncharged, and neutral quantum dotsthrough gold-coated pnc-Si membranes will be assessed using quantitativefluorescence microscopy. These experiments will establish baselinetransport through the gold-coated membranes. By attaching the coatedmembranes to a voltage source, the charge carried by the membranes willbe directly manipulated. It is expected to find slower transport ofnegatively charged species and that the transport rate varies inverselywith the voltage applied. As in method described for Experiment 1, theuse of quantum dots with neutralized charges will serve as controlsbecause their transport should be voltage independent. In addition,increasing the ionic strength of the solution should diminish theability of voltage to regulate the permeability of pnc-Si membranes tocharged species.

Experiment 3

In electrophoresis, a charged species is transported between electrodeswhen a voltage is applied across an electrolyte solution. Regardless ofthe charge on pnc-Si membranes, it should be possible to modulate thepassage of charged species through pnc-Si membranes by positioningelectrodes on either side of the membrane.

These experiments will again compare the transport of positive, neutral,and negatively charged quantum dots through (uncoated) pnc-Si membranesusing a microscope-based diffusion chamber, except that platinumelectrodes will be placed upstream and downstream and a voltage will beapplied to the solutions. Positive and negative quantum dots will beplaced together on one side of the membrane. Transport will be comparedfor voltages varying between 0 and 2 V. Bubbles caused by electrolysiscould easily block large sections of membrane and confound experiments,and so degassing solutions prior to the experiments and limitingvoltages to 2 V (Kuo et al., “Molecular Transport Through NanoporousMembranes,” Langmuir 17:6298-6303 (2001)) will help minimize theformation of oxygen bubbles. Additionally, one can conduct experimentswith the membrane vertical so that bubbles would be unlikely to remainproximal to the membranes. The expectation is that the rate of transportof either positive or negative species can be controlled by themagnitude and polarity of the applied voltage. Meanwhile the oppositelycharged species should migrate away from the membrane and thereby remaintrapped in the starting chamber. These experiments provide analternative to direct manipulation of membrane charge for controlledtransport past pnc-Si membranes.

Example 9 Modification of Dialysis Machine Using Porous NanocrystallineSilicon Membrane

The potential to use pnc-Si membranes in large-scale renal dialysis willbe examined with the expectation that the membranes will selectivelyremove small protein toxins (β₂ microglobulin) that currently causesignificant discomfort to patients undergoing dialysis treatments (Rajet al., “Beta(2)-microglobulin Kinetics in Nocturnal Haemodialysis,”Nephrol. Dial. Transplant 15(1):58-64 (2000), which is herebyincorporated by reference in its entirety). The mechanical strength ofpnc-Si membranes makes the construction of an assembly for large-scaleapplications like dialysis appear feasible.

A plurality of silicon wafers will be assembled into a flow-throughdevice where the membranes are characterized by maximal pore sizes ofabout 20 nm. When retrofitted onto to dialysis equipment (i.e.,replacing traditional dialysis tubing), blood flowing over the surfaceof the membrane will allow the small protein toxin and certain salts todiffuse across the membrane, thereby removing it from blood to bereturned to a patient.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method of producing a porous nanoscale membrane comprising:applying a nanoscale film to one side of a substrate, the nanoscale filmcomprising a semiconductor material; masking an opposite side of thesubstrate; etching the substrate, beginning from the masked oppositeside of the substrate and continuing until a passage is formed throughthe substrate, thereby exposing the film on both sides thereof to form amembrane; and converting the amorphous semiconductor material, before orafter said etching, to a crystalline semiconductor material and therebyforming a plurality of spaced pores in the membrane during saidconverting.
 2. The method according to claim 1 further comprising:applying a sacrificial film either (i) to the one side of the substrateprior to said applying the nanoscale film, (ii) over the nanoscale filmafter said applying the nanoscale film, or (iii) both (i) and (ii). 3.The method according to claim 2 wherein said applying the nanoscale filmand said applying the sacrificial film is carried out in a manner toform substantially planar films.
 4. The method according to claim 2wherein said applying the nanoscale film and said applying thesacrificial film is carried out in a manner to form a relief-patternedfilm.
 5. The method according to claim 2 wherein both (i) and (ii) arecarried out.
 6. The method according to claim 2 wherein the nanoscalefilm comprises an amorphous semiconductor material and each sacrificialfilm is an oxide film.
 7. The method according to claim 1 wherein saidconverting is carried out by annealing the amorphous semiconductormaterial at a temperature and duration suitable to inducecrystallization.
 8. The method according to claim 1 wherein thecrystalline semiconductor material is in the form of a polycrystallineor a nano crystalline material.
 9. The method according to claim 6wherein each oxide film is silicon dioxide and the substrate is silicon.10. The method according to claim 9 wherein said masking comprisesforming an incomplete silicon dioxide film on the opposite side of thesilicon substrate.
 11. The method according to claim 10 wherein saidetching is carried out using an etchant that has a silicon/silicondioxide selectivity that is greater than about 10,000.
 12. The methodaccording to claim 10 wherein said etching is carried out using anetchant comprising ethylenediamine, pyrocatechol, pyrazine, and water.13. The method according to claim 5 further comprising: removing thesacrificial films after said etching.
 14. The method according to claim13 wherein said removing is carried out after said converting.
 15. Themethod according to claim 13 wherein said removing comprises exposingthe sacrificial silicon dioxide films to a buffered oxide etchantsolution, or a hydrofluoric acid etchant solution.
 16. The methodaccording to claim 1 wherein said etching is carried out in a containerthat exposes substrate to etchant solution only from the masked oppositeside thereof.
 17. The method according to claim 1 further comprising:heating and/or stirring etchant solution during said etching.
 18. Themethod according to claim 1 wherein said applying comprisesradio-frequency magnetron sputtering, low pressure chemical vapordeposition, plasma enhanced chemical vapor deposition, thermal chemicalgrowth, radio frequency sputtering, DC sputtering, thermal evaporation,electron beam evaporation, or electroplating.
 19. The method accordingto claim 1 wherein said applying further comprises changing conditionsduring film deposition, the conditions being selected from the group ofpressure changes, plasma density changes, plasma power changes,temperature changes, source material changes, and gas compositionchanges.
 20. The method according to claim 1 wherein the semiconductormaterial is undoped silicon or germanium, p-doped silicon or germanium,n-doped silicon or germanium, or a silicon-germanium alloy.